Catalytic RNAs and retrotransposons are key RNA elements that help shape genomes. Bacterial group II introns (gII-ins) are both self-splicing RNAs, which are the putative progenitors of spliceosomal introns, and mobile retroelements, and as such they occupy a pivotal role in early eukaryotic evolution. At a structural level, gII-in RNA combines with an intron-encoded protein to form a ribonucleoprotein (RNP) that is active in both splicing and mobility. The overall goal of this application is to understand the structure and function of these bacterial mobile self-splicing retroelements, while relating them to their eukaryotic spliceosomal and retroelement counterparts. This goal will be achieved by combining interdisciplinary biochemical, biophysical, cellular, computational, genetic, structural and systems approaches. Considerable progress over the past funding period, including structural analysis of the gII-in RNP, and functional studies relating retrotransposition to conjugation, is te springboard for the proposed specific aims: Aim 1: To capture structure transitions of the gII-in RNP in splicing and gene targeting. We will use native RNPs purified from L. lactis, for which we have derived a 4.5 cryo-EM structure, to develop a series of structural and kinetic snapshots of the gII-in RNP at different stages. These include the loose RNP precursor; the compact, spliced free intron RNP; and the free intron RNP attacking its DNA target for retromobility. Emphasis will also be placed on modification of the intron RNA and its role in catalysis of splicing and retromobility, structure transitions and interactions with the intron-encoded protein. Finally, our recent discovery that the modular intron-encoded protein is similar to two pivotal eukaryotic proteins, Prp8, the most conserved protein in the spliceosome, and telomerase reverse transcriptase, which preserves chromosome ends, is the basis of tests of analogous functions in splicing and retromobility. Aim 2: To understand host-retrotransposon relationships across kingdoms. We will further investigate the common residence of gII-ins with other mobile elements, to determine their interrelationships with the bacterial mobilome. We will also define the dynamic relationship, both positive and negative, between the parasitic gII-in and the bacterial host, L. lactis, using biochemical, genetic and systems approaches. These studies will be integrated with the NIH-funded Center for Systems Biology of Retrotransposition, which focuses on mammalian retrotransposons. The impact of our interdisciplinary approach is an enhanced understanding of the structure and function of self-splicing elements that have been exploited biotechnologically to edit genes, and which resemble retrotransposons that sculpt diverse genomes in health and disease.